In vitro methods for determining in vivo thrombotic events

ABSTRACT

Diagnostic systems, methods, polypeptides and antibodies for detecting the presence of C-terminal hGPIIb fragment of the platelet receptor GPIIb-IIIa in a body fluid sample are disclosed.

This invention was made with government support under Grant No. GM 37696awarded by the National Institutes of Health.

This is a continuation of application Ser. No. 07/693,388 filed on Apr.30, 1991 now abandoned.

DESCRIPTION Technical Field

The present invention relates to a fragment of the C-terminal region ofGPIIb heavy chain (hGPIIb) that is produced in vivo by indigenousprotease cleavage of the GPIIb-IIIa platelet glycoprotein receptor, andto in vitro detection of the fragment in body fluids of individualsundergoing thrombotic events. The present invention also relates topolypeptide analogs of the hGPIIb C-terminal fragment, and antibodiesthat immunoreact with the C-terminal fragment and to diagnostic methodsfor detecting thrombi and clotting disorders.

Background

Thrombosis involves cell adhesion of platelets. Platelet cell adhesiongenerally involves recognition of specific adhesive proteins by theplatelet cell surface receptor GPIIb-IIIa.

GPIIb-IIIa is a noncovalent, Ca⁺⁺ dependent, heterodimer complexcomprised of noncovalently associated alpha and beta subunits. Jenningset al., J. Biol. Chem., 257:10458-10466 (1982). The alpha subunit, GPIIbconsists of a heavy chain (hGPIIb) having a relative molecular weight ofabout 120 kilodaltons (KDa), and a light chain (lGPIIb) of about 20 KDathat are linked together by disulfide bonds. The beta subunit, GPIIIa isa single chain polypeptide of about 100 KDa. Phillips et al., J. Biol.Chem., 252:2121-2126 (1977).

GPIIb-IIIa contributes to platelet function through interactions withRGD-containing proteins such as fibrinogen Bennett et al., Proc. Natl.Acad. Sci. USA, 80:2417-2421 (1983)!, fibronectin Ginsberg et al., J.Clin. Invest., 71:619-624 (1983)!, and von Willebrand factor Ruggeri etal., Proc. Natl. Acad. Sci. USA, 79:6038-6041 (1982)!, and therefore isa component of the common platelet adhesive protein receptor Pytela etal., Science, 231:1559-1562 (1986) and Plow et al., J. Biol. Chem.,259:5388-5391 (1984)!.

The sites on GPIIb-IIIa that are required to function as an adhesionreceptor are not well characterized. Several observations suggest that afunctionally significant site on GPIIb-IIIa is near the epitope definedby the monoclonal antibody PMI-1. This antibody binds to the heavy chainof GPIIb Shadle et al., J. Cell. Biol., 99:2056-2060 (1984)! and definesa region of GPIIb that is associated with several distinct functionalactivities. First, PMI-1 inhibits adhesion of washed platelets tocollagen. Shadle et al., J. Cell. Biol., 99:2056-2060 (1984). Second,the surface orientation of this region is regulated by divalent cationsbecause millimolar (mM) concentrations of calcium or magnesium suppressexpression of the PMI-1 epitope. Ginsberg et al., J. Clin. Invest.,78:1103-1111 (1986). Third, abnormal divalent cation regulation of theconformation of this site is associated with a functional thrombasthenicstate. Ginsberg et al., J. Clin. Invest., 78:1103-1111 (1986). Fourth,stimulation of platelets with up to 100 micromolar adenosine diphosphate(ADP) or epinephrine, 1 unit per milliliter thrombin, or 50 microgramsper milliliter calf skin collagen does not substantially increase thebinding of PMI-1 antibodies to platelets.

Platelet activation has been reported to produce the appearance ofantigenic sites on the platelet surface that are not present in thenon-activated platelet, and at least one of such induced sites has beenlocalized to the GPIIb-IIIa receptor complex. Shattil et al, J. Biol.Chem. 260:11107-11114 (1985); Coller, B. S., J. Cell Biol., 103:451-456(1986).

Numerous assays based on the release of fragments of blood coagulationenzymes or platelet activation have been developed in attempts toidentify thrombotic and pre-thrombotic states. From the plateletperspective, initial efforts were focused on release of plateletspecific antigens. Ginsberg, et al., In Immunologic Analysis: RecentProgress in Diagnostic Labor. Immunol., Nakamura et al., Eds. MassonPublishing, USA Inc., 199-209 (1982). Recent studies have focused on theuse of monoclonal antibodies selective for activated platelets.Frelinger et al., J. Biol. Chem., 263:12397-12402 (1988); Aiken et al.,Sem. Thromb. Hemost., 13:307-316 (1987); Berman et al., J. Clin.Invest., 78:130-137 (1986); Hsu-Lin et al., J. Biol. Chem.,259:9121-9126 (1984); George et al., J. Clin. Invest., 78:340-348(1986); Shattil et al., Blood, 73:150-158 (1989); Shattil et al., J.Biol. Chem., 260:11107-11114 (1985); Adelman et al., Blood, 70:1362-1366(1987).

Each of the above methods has been plagued by unintentional in vitroactivation because it is difficult to obtain blood samples in a mannersuch that the platelet activation events are not triggered duringvenipuncture, sample handling or the like. This difficulty has severelylimited the clinical utility of such assays. It would be desirable,therefore, to develop a method to detect the occurrence of thromboticevents without relying on platelet activation events. The utility ofdetermining the elevation in a plasma glycocalicin, as a potential assayfor in vivo platelet activation, has also been reported. Steinberg etal., N. Engl. J. Med., 317:1037-1042 (1987); Coller et al., J. Clin.Invest., 73:794-799 (1984).

It has recently been found that a class of antigenic determinants areexpressed when GPIIb-IIIa specifically binds to its ligand. Theantigenic determinants are not expressed by either the non-occupiedGPIIb-IIIa receptor or the non-bound ligand. One such determinantlocated on the C-terminus of the GPIIB heavy chain is recognized by thepreviously described PMI-1 monoclonal antibody. Shadle et al., J. CellBiol., 99:2056-2060 (1984); Frelinger et al., J. Biol. Chem.,263:12397-12402 (1988).

BRIEF SUMMARY OF THE INVENTION

It has now been found that a portion of the C-terminus of hGPIIb,containing the PMI-1 antigenic determinant, becomes exposed uponreceptor-ligand binding and is cleaved by indigenous proteases inducedby thrombus formation to form a cleaved fragment. The fragment, referredto as C-terminal hGPIIb fragment, is released into the vascular fluid,and finds its way into other body fluids, such as urine.

Simple activation of platelets with agonists in vitro is not sufficientto release the C-terminal hGPIIb fragment. Treatment with a protease isrequired. In the thrombus, the platelet receptor is occupied, therebyexposing this region of the molecule, and a variety indigenousproteases, such as plasmin, are generated. Thus, in the presentinvention, detecting the release of C-terminal hGPIIb fragment byproteases is not subject to the problems of in vitro activation, and canbe performed on a routinely obtained plasma or urine specimen from apatient.

The principal utility of the present invention is its ability toidentify in vitro thrombotic events ongoing in vivo, and to monitorthrombolysis. Clinical settings include acute thrombotic events such asstroke or coronary thrombosis, or more chronic events such as deepvenous thrombosis. In addition, detection of chronic ongoing thromboticevents can serve to identify individuals at risk of developing acutethrombosis.

The present invention is directed to a substantially isolated C-terminalhGPIIb fragment that has a molecular weight of about 3900 daltons,comprising an amino acid residue sequence that corresponds to thecarboxy terminal portion of the amino acid residue sequence of hGPIIbshown in SEQ ID NO 3, including the amino acid residue sequencerepresented by the formula --IHPAHHK--, shown in SEQ ID NO 3 fromresidue 194 to residue 200. This fragment has the capacity toimmunoreact with the monoclonal antibody PMI-l, and preferably has theamino acid residue sequence comprising the amino acid residue sequenceshown in SEQ ID NO 3 from about residue 173 to about residue 200.

Also contemplated is a hGPIIb analog comprising a polypeptide of no morethan about 200 residues, having an amino acid residue sequence thatcorresponds to the sequence of hGPIIb shown in SEQ ID NO 3 and includesat least about 7 contiguous amino acid residues from the amino acidresidue sequence of hGPIIb shown in SEQ ID NO 3 from residue 173 toresidue 200. A preferred analog is a polypeptide comprising an aminoacid residue sequence corresponding to the formula: --PQPPVNPLK--;--PQPPVNLPLKVDWGLPIPSPSP--; --PLKVDWGLPIP--; --PIPSPSPIHPAHHK--; or--IHPAHHK-- having a sequence shown in SEQ ID NO 3 from residue 173 toresidue 181, from residue 173 to residue 193, from residue 179 toresidue 189, from residue 187 to residue 200, or from residue 194 toresidue 200.

Further contemplated is an antibody comprising an antibody molecule orfragment thereof that is capable of immunoreacting specifically withC-terminal hGPIIb fragment but does not substantially immunoreact with apolypeptide represented by the formula REQNSLDSWGPK having the sequencein SEQ ID NO 1 from residue 113 to residue 124. In preferredembodiments, the antibody composition contains a monoclonal antibody orfragment thereof that is substantially similar to the antibody, orfragment of the antibody, produced by hybridoma PMI-1.

The present invention also contemplates a method for assaying thepresence of a C-terminal hGPIIb fragment in a body fluid sample. Themethod comprises admixing an aliquot of body fluid with an antibody ofthis invention. Contact of the body fluid and the antibody is maintainedfor a time period sufficient for an immunoreaction product to form. Thefragment-containing immunoreaction product formed is then detected,thereby indicating the presence of the C-terminal hGPIIb fragment in thesample.

A diagnostic system in kit form is further contemplated by the presentinvention. In one embodiment is a diagnostic system in kit form forassaying for the presence of a C-terminal hGPIIb fragment in a bodyfluid sample. The system comprises a package containing, in an amountsufficient to perform at least one assay, an antibody of this invention.The antibody can be in a liquid solution but in one embodiment ispreferably attached to a solid-phase matrix. The system may also containa label for indicating the presence of the antibody molecules in theimmunoreaction product formed.

In a more preferred embodiment, the diagnostic system further contains asolid support comprising a solid matrix having affixed thereto at leastone of the set of hGPIIb analog and a polypeptide having an amino acidresidue sequence that includes the sequence of a C-terminal hGPIIbfragment, thereby providing a kit for assaying by a competition ELISA avascular fluid or urine sample for the presence of C-terminal hGPIIbfragment.

In one preferred embodiment, the before-described method is utilized toassess the extent of acute thrombosis by detecting the presence ofC-terminal hGPIIb fragment in a body fluid sample and relating theamount of immunoreaction product formed to predetermined standardscorrelative with the diseased state.

In another preferred embodiment, this method is useful to monitor thecourse of plasminogen activator-induced thrombolysis by detecting thepresence of C-terminal hGPIIb fragment in a body fluid sample andrelating the amount to correlative predetermined levels.

In yet another preferred embodiment, this method is contemplated foridentifying individuals at risk of developing acute thrombosis, whichmethod determines the extent of chronic thrombosis and relates it topredetermined levels correlating with the risk of acute thrombosis.

BRIEF DESCRIPTION OF THE DRAWINGS

In the Figures forming a portion of the disclosure of this invention:

FIGS. 1A and 1B illustrates the effect of in vitro incubation ofplatelets at 37C. in the presence of 30 uM chymotrypsin as described inExample lc. One portion of the digested platelet suspension wascentrifuged and the supernatant (open diamond) and resuspended pellet(+) were assayed separately. An equal portion of the platelet suspensionwas assayed comprising total PMI-1 immunoreactive material (opensquare). Levels of the PMI-1 epitope (FIG. 1A) or the Tab epitope (FIG.1B) (a monoclonal antibody with a different GPIIb epitope specificity)were determined by competitive ELISA assays, as described hereinafter.Note the release of material reactive with PMI-1, but not with Tab, intothe platelet-free supernatant.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Amino Acid: An amino acid formed upon chemical digestion (hydrolysis) ofa polypeptide at its peptide linkages. The amino acid residuesidentified herein are preferably in the natural "L"isomeric-configuration. However, residues in the "D" isomeric form canbe substituted for any L-amino acid residue, as long as the desiredfunctional property is retained by the polypeptide. NH₂ refers to thefree amino group present at the amino terminus of a polypeptide. COOHrefers to the free carboxy group present at the carboxy terminus of thepolypeptide. In keeping with standard polypeptide nomenclature, J. Biol.Chem., 243:3557-59 (1969) and adopted at 37 C.F.R. 1.822(b)(2),abbreviations for amino acid residues are as shown in the followingTable of

    ______________________________________                                        TABLE OF CORRESPONDENCE                                                       SYMBOL                                                                        1-Letter      3-Letter                                                                              AMINO ACID                                              ______________________________________                                        Y             Tyr     L-tyrosine                                              G             Gly     glycine                                                 F             Phe     L-phenylalanine                                         M             Met     L-methionine                                            A             Ala     L-alanine                                               S             Ser     L-serine                                                I             Ile     L-isoleucine                                            L             Leu     L-leucine                                               T             Thr     L-threonine                                             V             Val     L-valine                                                P             Pro     L-proline                                               K             Lys     L-lysine                                                H             His     L-histidine                                             Q             Gln     L-glutamine                                             E             Glu     L-glutamic acid                                         W             Trp     L-tryptophan                                            R             Arg     L-arginine                                              D             Asp     L-aspartic acid                                         N             Asn     L-asparagine                                            C             Cys     L-cysteine                                              ______________________________________                                    

It should be noted that all amino acid residue sequences are representedherein by having a left to right orientation in the conventionaldirection of amino-terminus to carboxy-terminus. In addition, the phase"amino acid residue" is broadly defined to include modified and unusualamino acids, such as those listed in 37 C.F.R. 1.822(b)(4), and areincorporated by reference. Furthermore, it should be noted that a dashat the beginning or end of an amino acid residue sequence indicates apeptide bond to a further sequence of one or more amino acid residues ora covalent bond to an amino-terminal group such as NH₂ or acetyl or to acarboxy-terminal group such as COOH.

Polypeptide and Peptide: Polypeptide and peptide are terms usedinterchangeably herein to designate a linear series of no more thanabout 50 amino acid residues connected one to the other by peptide bondsbetween the alpha-amino and carboxy groups of adjacent residues.

Protein: Protein is a term used herein to designate a linear series ofgreater than 50 amino acid residues connected one to the other as in apolypeptide.

II. Introduction

Platelets play a central role in thrombosis. The invention provides anin vitro means to detect platelet incorporation into thrombi in vivo,and thereby detects thrombotic events.

When platelets are incorporated into a thrombus, occupancy of GPIIb-IIIareceptor by its ligand is believed to expose the C-terminal region ofthe GPIIb heavy chain. Indigenous proteases, such as plasmin orleukocyte enzymes, generated at the site of the thrombus in vivo cleavethe C-terminal portion of hGPIIb, releasing the small peptide fragmentsdescribed above. Measurement of these fragments in vascular fluid or inurine provides a diagnostic correlate of the extent of the thromboticprocess. These measurements are also valuable in monitoring the courseof thrombolysis induced by plasminogen activators.

PMI-1, a monoclonal antibody derived from immunization of mice withisolated human platelet plasma membranes, reacts with the carboxyterminal aspect of the heavy chain of the platelet membrane glycoproteinhGPIIb. Shadle et al., J. Cell Biol., 99:2056-2060 (1984); and Loftus etal., Proc. Natl. Acad. Sci. USA, 840:7114-7118 (1987). In the presentinvention, an enzyme-linked immunosorbent assay (ELISA) has beendeveloped to measure levels of PMI-1 antigenic material. The principalobservation, illustrated in FIG. 1, is that in vitro treatment ofplatelets with protease releases from the carboxy terminus of the GPIIbheavy chain a small (approximately 7 kDa, based on average MW of aminoacid residue=120) fragment that contains the PMI-1 epitope. The fragmentis referred to herein as the C-terminal hGPIIb fragment.

A potential site of cleavage of GPIIb-IIIa by indigeneous proteases isdownstream from the cysteine residue at position 171 of the sequenceshown in SEQ ID NO 3. The cysteine at position 171 participates indisulfide bridging to the light chain of GPIIb. Calvette et al.,Biochem. J., 261:551-560 (1989). The preferred site for chymotrypsincleavage of GPIIb-IIIa when present on platelets is between residues 172and 173 of the sequence shown in SEQ ID NO 3.

The carboxy terminus of hGPIIb has been identified by Loftus et al., J.Biol. Chem. 263:11025 (1988). After initial processing to cleave theGPIIb precursor protein into the heavy and light chain, alternateproteolytic processing results in partial trimming back of the carboxyterminus of hGPIIb. The partial proteolysis produces a polypeptidehaving either an lysine (K) or arginine (R) at the carboxy terminus,corresponding to residue 200 or 201, respectively, of the sequence shownin SEQ ID NO 3.

The capacity of the PMI-1 monoclonal antibody to inhibit plateletadhesion to collagen, Shadle et al., J. Cell Biol., 99:2056-2060 (1984),its upregulation by chelation of divalent cations, Ginsberg et al., J.Clin. Invest., 78:1103-1111 (1986), and by occupancy of GPIIb-IIIa withadhesive ligands, Frelinger et al., J. Biol. Chem., 263:12397-12402(1988), has been previously published by the applicants and is wellknown. There have, however, been no prior publications on the use of anELISA technique for detecting this novel hGPIIb fragment as a means ofidentifying and evaluating thrombosis.

As discussed before, and reviewed by Ginsberg et al., in ImmunologicAnalysis: Recent Progress in Diagnostic Labor. Immunol., Nakamura etal., Eds. Masson Publishing, USA Inc., 199-209 (1982), there have beennumerous assays designed to quantitate released platelet granulecomponents in the circulation as a means for detecting plateletactivation. The present invention, however, does not involve release ofa granule component, but rather involves the detection of a proteolyticattack on a platelet surface component, and as such is less susceptibleto in vitro artifact, e.g., false positive results due to in vitroactivation of platelets.

III. hGPIIb Fragment and Analogs

A. Fragment

In one embodiment, the present invention contemplates a substantiallyisolated C-terminal hGPIIb fragment having a molecular weight of about3900 daltons, comprising an amino acid residue sequence that correspondsto the carboxy-terminal portion of the amino acid residue sequence ofhGPIIb. The C-terminal hGPIIb fragment includes as a portion of itsamino acid residue sequence the sequence represented by the formula--IHPAHHK-- shown is SEQ ID NO 3 from residue 194 to residue 200, andhas the capacity to immunoreact with the monoclonal antibody, PMI-1.Preferably, the C-terminal hGPIIb fragment has the amino acid residuesequence shown in SEQ ID NO 3 from about residue 173 to about residue200.

A C-terminal hGPIIb fragment can be purified from human urine or plasmausing well known immunoaffinity methods, such as described in Example1c(2). Alternatively, C-terminal GPIIb fragment can be produced in vitroby proteolysis of platelets or by proteolysis of isolated GPIIb-IIIa,and subsequently immunoaffinity purified as described in Example 1.Proteolysis of platelets or isolated GPIIb-IIIa using the proteasechymotrypsin is particularly preferred. The latter procedure ispreferred due to the availability of platelets and the amount ofGPIIb-IIIa contained therein.

C-terminal hGPIIb fragment is substantially isolated if compositionscontaining the fragment are comprised of greater than 5 percent, byweight, of fragment per total protein in the composition.

C-terminal hGPIIb fragment, once isolated, is useful to produceimmunogen for raising antibody specific for the fragment, and for use inthe diagnostic methods and systems for detecting the presence and amountof C-terminal hGPIIb fragment in body fluid samples, as disclosedherein.

B. Polypeptide Analogs of the C-Terminal hGPIIb Fragment

One embodiment of the present invention contemplates a polypeptide thatincludes amino acid residue sequences capable of mimicking antigenicdeterminants expressed by the C-terminal hGPIIb fragment. The hGPIIbfragment-mimicking polypeptide is also referred to herein as an analog,or hGPIIb analog. A hGPIIb polypeptide analog of the present inventioncomprises an amino acid residue sequence that corresponds, andpreferably is identical, to a portion of the hGPIIb sequence shown inSEQ ID NO 1 from residue 1 to residue 200. The hGPIIb analog is fromabout 7 to about 200 amino acid residues in length, preferably less thanabout 50, and more preferably less than 20 residues in length, andincludes a minimum of about 7 contiguous residues of the hGPIIb aminoacid residue sequence shown in SEQ ID NO 3 from residue 173 to residue200.

In one preferred embodiment, a hGPIIb analog includes at least thefollowing amino acid residue sequence:

    --IHPAHHK--,

representing the amino acid residues 194-200 shown in SEQ ID NO 3. Inthis embodiment, a hGPIIb analog is also capable of immunoreacting withthe monoclonal antibody, PMI-1. The antibody PMI-1 is known to beimmunospecific for (i.e., immunoreacts with) the minimum epitope--IHPAHHK-- on hGPIIb.

In another embodiment, a preferred hGPIIb analog is a polypeptide thatcomprises an amino acid residue sequence corresponding to one of thefollowing amino acid sequences:

    --PQPPVNPLK--,

    --PLKVDWGLPIP--,

    --PIPSPSPIHPAHHK--, or

    --IHPAHHK--,

having an amino acid residue sequence shown in SEQ ID NO 3 from residue173 to residue 181, from residue 179 to residue 189, from residue 187 toresidue 200, or from residue 194 to residue 200, respectively.

Particularly preferred hGPIIb analogs are contemplated having an aminoacid residue sequence as shown in Table I below.

                  TABLE 1                                                         ______________________________________                                        Designation.sup.a                                                                            Amino Acid Residue Sequence                                    ______________________________________                                        p173-193       PQPPVNPLKVDWGLPIPSPSP                                          p173-181       PQPPVNPLK                                                      p179-189       PLKVDWGLPIP                                                    p187-200       PIPSPSPIHPAHHK                                                 p194-200       IHPAHHK                                                        ______________________________________                                         .sup.a The designation of each polypeptide represents the included amino      acid residue sequence as shown in SEQ ID NO 3.                           

One contemplated hGPIIb analog has an amino acid residue sequence thatcorresponds to the sequence shown in SEQ ID NO 3 from residue 1 toresidue 200. More preferred is a hGPIIb analog that corresponds insequence to a C-terminal hGPIIb fragment and has a sequence shown in SEQID NO 3 from residue 173 to residue 200.

The polypeptides shown in Table 1 are further characterized by theirability to neutralize (competitively inhibit) the binding of antibodymolecules to their respective antigenic determinants on the nativehGPIIb molecule, described hereinbelow.

It should be understood that a hGPIIb analog of the present inventionneed not be identical to the amino acid residue sequence of hGPIIb, solong as it has sufficient similarity with (i.e., corresponds to)portions of hGPIIb that the analog is able to competitively inhibit theimmunoreaction of specific antibody molecules, elicited as describedhereinbelow. Therefore, a hGPIIb analog can be subject to variouschanges, such as insertions, deletions and substitutions, eitherconservative or non-conservative, where such changes provide for certainadvantages in their use.

Conservative substitutions are those where one amino acid residue isreplaced by another, biologically similar residue. Examples ofconservative substitutions include the substitution of one hydrophobicresidue, such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as betweenarginine and lysine, between glutamic and aspartic acids or betweenglutamine and asparagine and the like. The term "conservativesubstitution" also includes the use of a substituted amino acid in placeof an unsubstituted parent amino acid provided that such a polypeptidealso displays the requisite immunoreaction activity.

When a polypeptide of the present invention has a sequence that is notidentical to the corresponding sequence of hGPIIb because one or moreconservative or non-conservative substitutions have been made, usuallyno more than about 20% and preferably no more than 10% of the amino acidresidues are substituted, except where additional residues have beenadded at either terminus for the purpose of providing a "linker" bywhich the polypeptides of this invention can be conveniently affixed toa label or solid matrix, or antigenic carrier. Labels, solid matricesand carriers that can be used with the polypeptides of this inventionare described hereinafter.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues. Typical amino acidresidues used for linking are tyrosine, cysteine, lysine, glutamic andaspartic acid, or the like. In addition, a polypeptide sequence of thisinvention can differ from the natural sequence by the sequence beingmodified by terminal-NH₂ acylation, e.g., acetylation, or thioglycolicacid amidation, terminal-carboxlyamidation, e.g., ammonia, methylamine,etc.

When coupled to a carrier via a linker to form what is known in the artas a carrier-hapten conjugate, a hGPIIb analog of the present inventionis capable of inducing formation of antibodies that immunoreact with theC-terminal fragment of hGPIIb when present as a free-floating fragmentin vascular fluid or urine.

In view of the well established principle of immunologiccross-reactivity, the present invention therefore contemplatesantigenically related variants of the polypeptides described before. An"antigenically related variant" is a polypeptide that is capable ofinducing formation of antibody molecules that immunoreact with a hGPIIbanalog and with the C-terminal hGPIIb fragment when present in bodyfluids.

Any hGPIIb analog of the present invention can be synthesized by one ofmany techniques known to those skilled in the polypeptide art, includingrecombinant DNA techniques. Synthetic chemistry techniques, such as asolid-phase Merrifield-type synthesis, are preferred for reasons ofpurity, antigenic specificity, freedom from undesired side products,ease of production and the like. An excellent summary of the manytechniques available can be found in J. M. Steward and J. D. Young,"Solid Phase Peptide Synthesis", W. H. Freeman Co., San Francisco(1969); Bodansky et al., "Peptide Synthesis", John Wiley & Sons, 2ndEd., (1976); and Meienhofer, "Hormonal Proteins and Peptides", 2:46,Academic Press, NY (1983) for solid phase peptide synthesis; andSchroder et al., "The Peptides", Vol. 1, Academic Press, NY (1965) forclassical solution synthesis, each of which is incorporated herein byreference. Appropriate protective groups usable in such synthesis aredescribed in the above texts and in Mcomie, "Protective Groups inOrganic Chemistry", Plenum Press, NY (1973), which is incorporatedherein by reference.

In general, the solid-phase synthesis methods contemplated comprise thesequential addition of one or more amino acid residues or suitablyprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

Using a solid-phase synthesis as exemplary, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting groups (and solid support) are removedsequentially or concurrently, to afford the final polypeptide.

A hGPIIb analog can be used, inter alia, in the diagnostic methods andsystems of the present invention to detect C-terminal hGPIIb fragmentpresent in a body s am ple, or can be used to prepare an inoculum asdescribed herein for the preparation of antibodies that immunoreact withthe fragment.

IV. Antibodies and Antibody Compositions

The term "antibody" in its various grammatical forms is used herein torefer to immunoglobulin molecules and fragments thereof, namelyimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antibody combining site or paratope.Illustrative antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contain the paratope, including thoseportions known in the art as Fab, Fab', F(ab')₂ and F(v).

The term "antibody combining site" refers to that structural portion ofan an tibody molecule, comprised of a heavy and light chain variable andhypervariable regions, that specifically binds (immunoreacts with) anantigen. The term "immunoreact" in its various forms is used herein torefer to binding between an antigenic determinant-containing moleculeand a molecule containing an antibody combining site such as a wholeantibody molecule or a portion thereof.

The term "antigenic determinant" refers to the actual structural portionof the antigen that is immunologically bound by an antibody combiningsite. The term is also used interchangeably with "epitope".

As used herein, the term "specifically bound" refers to a non-randombinding reaction between an antibody and a antigen.

A. Antibody Compositions

An antibody composition of the present invention is characterized ascontaining antibody molecules that immunoreact with a C-terminal hGPIIbfragment or hGPIIb analog as described herein.

An antibody of this invention, i.e., an anti-C-terminal hGPIIb fragmentantibody, does not immunoreact with other portions of the hGPIIbmolecule, such as antigenic determinants defined by polypeptidescorresponding in sequence to regions of hGPIIb that do not includeresidues 173 through 200 of the sequence shown in SEQ ID NO 3. Thus, forexample, a contemplated antibody does not immunoreact with a polypeptidehaving a sequence represented by the formula REQNSLDSWGPK, shown in SEQID NO 3 from residue 113 to residue 124. That is, the antibody isspecific for the C-terminal hGPIIb fragment identified and definedherein.

Thus an antibody, in its various embodiments, immunoreacts with anepitope present on a C-terminal hGPIIb fragment. Exemplary epitopes aredefined by the C-terminal hGPIIb analogs designated pl73-181, pl79-189,pl87-200 or pl94-200. Additional epitopes can reside within the hGPIIbC-terminal fragment that are useful for defining the immunoreactivity ofan antibody of this invention. Thus other embodiments contemplate anantibody that immunoreacts with a polypeptide defining regions otherthan the pl94-200 portion of hGPIIb C-terminal fragment. Exemplaryantibodies immunoreact with the polypeptide pl73-193, shown in SEQ ID NO3 from residues 173 to 193.

Such a contemplated antibody composition is typically produced byimmunizing a mammal with an inoculum containing a C-terminal hGPIIbfragment of this invention, or a polypeptide analog, thereby inducingproduction in the mammal of antibody molecules having immunospecificityfor the C-terminal hGPIIb fragment. The antibody molecules are thencollected from the mammal and isolated to the extent desired by wellknown techniques such as, for example, by using DEAE Sephadex to obtainthe IgG fraction. To enhance the specificity of the antibody, theantibodies may be purified by immunoaffinity chromatography using solidphase-affixed immunizing polypeptide or C-terminal hGPIIb fragment. Theantibody is contacted with the solid phase-affixed immunizingpolypeptide or fragment (immunogen) for a period of time sufficient forthe immunogen to immunoreact with the antibody molecules to form a solidphase-affixed immunocomplex. The bound antibodies are separated from thecomplex by standard techniques.

Preferred and exemplary are the methods to produce and isolateantibodies described in Example 3.

The antibody composition so produced can be used inter alia, in thediagnostic methods and systems of the present invention to detect thepresence and/or amount of C-terminal hGPIIb fragment in a body sample.

A preferred antibody in an antibody composition of this invention is amonoclonal antibody.

B. Inocula

The word "inoculum" in its various grammatical forms is used herein todescribe a composition containing a polypeptide or the C-terminal hGPIIbfragment of this invention as an active ingredient used for thepreparation of antibodies that immunoreact with the C-terminal hGPIIbfragment. The inoculum also contains a pharmaceutically acceptableaqueous diluent such that, when administered by immunization, is capableof eliciting antibodies that immunoreact with the hGPIIb C-terminalfragment.

When a polypeptide is used to induce antibodies it is to be understoodthat the polypeptide can be used alone, or linked to a carrier as aconjugate, or as a polypeptide polymer, but for ease of expression, thevarious embodiments of the polypeptides of this invention arecollectively referred to herein by the term "polypeptide", and itsvarious grammatical forms.

For a polypeptide that contains fewer than about 35 amino acid residues,it is preferable to use the peptide bound to a carrier for the purposeof inducing the production of antibodies as already noted.

As discussed before, one or more additional amino acid residues can beadded to the amino- or carboxy-termini of the polypeptide to assist inbinding the polypeptide to a carrier. Cysteine residues added to theamino- or carboxy-termini of the polypeptide have been found to beparticularly useful for forming conjugates via disulfide bonds. However,other methods well known in the art for preparing conjugates can also beused. Exemplary additional linking procedures include the use of Michaeladdition reaction products, di-aldehydes such as glutaraldehyde,Klipstein et al., J. Infect. Dis., 147, 318-326 (1983) and the like, orthe use of carbodiimide technology, as in the use of a water-solublecarbodiimide to form amide links to the carrier. For a review of proteinconjugation or coupling through activated functional groups, seeAvrameas, et al., Scand. J. Immunol., 1:7-23 (1978).

Useful carriers are well known in the art, and are usually proteinsthemselves. Examples of such carriers are keyhole limpet hemocyanin(KLH), edestin, thyroglobulin, albumins, such as bovine serum albumin(BSA) or human serum albumin (HSA), red blood cells, such as sheeperythrocytes (SRBC), tetanus toxoid, cholera toxoid as well as polyaminoacids, such as poly (D-lysine: D-glutamic acid), and the like.

The choice of carrier is dependent upon the ultimate use of the inoculumand is based upon criteria not particularly involved in the presentinvention. For example, a carrier that does not generate an undesiredreaction in the particular animal to be inoculated should be selected.

The inoculum contemplated herein contains an effective immunogenicamount of an immunogen of this invention, e.g., an hGPIIb analog or aC-terminal hGPIIb fragment, typically as a conjugate linked to acarrier. The effective amount of polypeptide or protein per unit dosesufficient to induce an immune response to the immunogen depends, amongother things, on the species of animal inoculated, the body weight ofthe animal and the chosen inoculation regimen as is well known in theart. Inocula typically contain polypeptide or protein concentrations ofabout 10 micrograms to about 500 milligrams per inoculation (dose),preferably about 50 micrograms to about 50 milligrams per dose.

The term "unit dose" as it pertains to the inocula of the presentinvention refers to physically discrete units suitable as unitarydosages for animals, each unit containing a predetermined quantity ofactive material calculated to produce the desired immunogenic effect inassociation with the required diluent; i.e., carrier, or vehicle. Thespecifications for the novel unit dose of an inoculum of this inventionare dictated by and are directly dependent on (a) the uniquecharacteristics of the active material and the particular immunologiceffect to be achieved, and (b) the limitations inherent in the art ofcompounding such active material for immunologic use in animals, asdisclosed in detail herein, these being features of the presentinvention.

Inocula are typically prepared from the dried solidpolypeptide-conjugate by dispersing the polypeptide-conjugate in aphysiologically tolerable (acceptable) diluent or vehicle such as water,saline or phosphate-buffered saline to form an aqueous composition. Suchdiluents are well known in the art and are discussed, for example, inRemington's Pharmaceutical Sciences, 16th Ed., Mack Publishing Company,Easton, PA (1980) at pages 1465-1467.

Inocula can also include an adjuvant as part of the diluent. Adjuvantssuch as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant(IFA) and alum are materials well known in the art, and are availablecommercially from several sources.

C. Monoclonal Antibody Compositions

The phrase "monoclonal antibody composition" in its various grammaticalforms refers to a population of antibody molecules that contain only onespecies of antibody combining site capable of immunoreacting with aparticular antigen. A monoclonal antibody composition thus typicallydisplays a single binding affinity for any antigen with which itimmunoreacts. A monoclonal antibody composition is typically composed ofantibodies produced by clones of a single cell called a hybridoma thatsecretes (produces) but one kind of antibody molecule. The hybridomacell is formed by fusing an antibody-producing cell and a myeloma orother self-perpetuating cell line. Such antibodies were first describedby Kohler and Milstein, Nature 256:495-497 (1975), which description isincorporated by reference. Detailed preparation of a monoclonal antibodycomposition is described further herein.

Thus, a monoclonal antibody of this invention immunoreacts with aC-terminal hGPIIb fragment or hGPIIb analog of this invention, but doesnot substantially immunoreact with a polypeptide having a sequencerepresented by the formula REQNSLDSWGPK shown in SEQ ID NO 3 fromresidue 113 to residue 124. That is, the antibody is specific for theC-terminal hGPIIb fragment.

Particularly preferred is a monoclonal antibody that immunoreacts with ahGPIIb analog of this invention, particularly the polypeptide IHPAHHK orpolypeptides that contain the sequence --IHPAHHK-- shown in SEQ ID NO 3from residues 194 to 200. Exemplary of this preferred embodiment is themonoclonal antibody PMI-1, available from the American Type CultureCollection (ATCC, Rockville, Md.) and having ATCC accession number HB9476, that has been shown to be immunospecific for an epitope on hGPIIbdefined by the polypeptide IHPAHHK and present on a C-terminal hGPIIbfragment.

In a related embodiment a monoclonal antibody is contemplated thatimmunoreacts with a hGPIIb analog defined by a polypeptide comprising asequence of amino acid residues shown in SEQ ID NO 3 from residues 173to 193, particularly a polypeptide having the sequence shown in SEQ IDNO 3 from residues 173 to 181, from residues 179 to 189, or fromresidues 179 to 193.

V. Methods for Producing Monoclonal Antibody Compositions

The present invention contemplates a method of forming a monoclonalantibody molecule that (a) immunoreacts with an antigenic determinant(epitope) on the C-terminal hGPIIb fragment, said fragment including anamino acid residue sequence as shown in SEQ ID NO 3 from residues 173 to200, from residues 173 to 181, from residues 173 to 193, from residues179 to 189, from residues 179 to 193, or from residues 194 to 200, but(b) does not substantially immunoreact with a polypeptide represented bythe formula REQNSLDSWGPK shown in SEQ ID NO 3 from residues 113 to 124.

A preferred method comprises the steps of:

(a) Immunizing an animal with an inoculum containing a C-terminal hGPIIbfragment or a polypeptide hGPIIb analog as described herein as theimmunogen. Preferably, the immunogen is complexed with a carrier orimmunopotentiator such as an ISCOM particle, liposome or the like. Theinoculum is administered to an immunologically competent mammal in anamount sufficient to produce an immune response. Preferably, the mammalis a rodent such as a rabbit, rat or mouse. The mammal is thenmaintained for a time period sufficient for it to produce cellssecreting antibody molecules that immunoreact with the C-terminal hGPIIbfragment.

(b) Preparing a suspension of immune cells. This is typicallyaccomplished by removing the spleen of the mammal and mechanicallyseparating the individual spleen cells in a physiologically tolerablemedium using methods well known in the art.

(c) Treating the suspended antibody-producing cells with a transformingagent capable of producing a transformed ("immortalized") cell line.Transforming agents and their use to produce immortalized cell lines arewell known in the art and include DNA viruses such as Epstein Bar Virus(EBV), Simian Virus 40 (SV40), Polyoma Virus and the like, RNA virusessuch as Moloney Murine Leukemia Virus (Mo-MuLV), Rous Sarcoma Virus andthe like, myeloma cells such as P3X63-Ag8.653, Sp2/O-Ag14, that areavailable from the ATCC under the designations CRL 1580 and CRL 1581,respectively, and the like.

In preferred embodiments, treatment with the transforming agent resultsin the production of a hybridoma by means of fusing the suspended spleencells with mouse myeloma cells from a suitable cell line by the use of asuitable fusion promoter. The preferred ratio is about 5 spleen cellsper myeloma cell in a suspension containing about 108 splenocytes. Apreferred fusion promoter is polyethylene glycol having an averagemolecule weight from about 1000 to about 4000 (commercially available asPEG 1000, etc.); however, other fusion promoters known in the art can beemployed.

The cell line used should preferably be of the so-called "drugresistant" type, so that unused myeloma cells will die in a selectivemedium, whereas hybrids will survive. The most cell lines used for thispurpose are 8-azaguanine resistant. These lines lack the enzymehypoxanthine-guanine phosphoribosyl transferase and hence will not besupported by HAT (hypoxanthine, aminopterin, and thymidine) medium. Itis also generally preferred that the myeloma cell line used be of theso-called "non-secreting" type, i.e., does not itself produce antibodymolecules. In certain cases, however, secreting myeloma lines may bepreferred.

(d) Cloning the transformed cells, preferably to monoclonality. Thecloning is preferably performed in a tissue culture medium that will notsustain (support) non-transformed cells. When the transformed cells arehybridomas, this is typically performed by diluting and culturing inseparate containers the mixture of unused spleen cells, unused myelomacells, and fused cells (hybridomas) in a selective medium which will notsustain the unused myeloma cells. The cells are cultured in this mediumfor a time sufficient to cause death of the unused cells (about oneweek). The dilution can be a limiting dilution, in which the volume ofdiluent is statistically calculated to isolate a certain number of cells(e.g., 1-4) in each separate container (e.g., each well of a microtiterplate). The medium is one (e.g., HAT medium) that will not sustain thedrug-resistant (e.g., 8-azaguanine resistant) unused myeloma cell line.

(e) Screening the tissue culture medium of the cloned transformants todetect the presence of secreted antibody molecules as discussed inExample 4a. Preferably, the medium is monitored using well knownimmunological techniques for the appearance of antibody molecules thatimmunoreact with C-terminal hGPIIb fragment.

(f) Selecting a desired transformant and growing it in an appropriatetissue culture medium for a suitable length of time, and harvesting theantibody from the culture supernatant by well known techniques. Thesuitable medium and suitable length of culturing time are also wellknown or are readily determined.

A monoclonal antibody of the present invention can also be produced bypreparing a culture containing a monoclonal hybridoma that secretesantibody molecules of the appropriate polypeptide specificity. Theculture is maintained under conditions and for a time period sufficientfor the hybridoma to secrete the antibody molecules into the medium. Theantibody-containing medium is then collected. The antibody molecules canthen be further isolated by well known techniques.

Representative and preferred methods for producing monoclonal antibodycompositions of this invention are described in Example 3c.

To produce a much greater concentration of slightly less pure monoclonalantibody, the selected hybridoma can be transferred by injection intomice, preferably syngeneic or semisyngeneic mice. The hybridoma willcause formation of antibody-producing ascites tumors after a suitableincubation time, which will result in a high concentration of thedesired antibody (about 5-20 mg/ml) in the bloodstream and peritonealexudate of the host mouse.

Media and animals useful for the preparation of these compositions areboth well known in the art and commercially available and includesynthetic culture media, inbred mice and the like. One commonly usedsynthetic medium is Dulbecco's minimal essential medium DMEM; Dulbeccoet al., Virol. 8:396 (1959)! supplemented with 4.5 gm/l glucose, 20 mMglutamine, and 20% fetal calf serum. A typical inbred mouse strain isthe Balb/c.

The monoclonal antibody compositions produced by the above method can beused in the same manner as disclosed herein for antibodies of thisinvention, and are preferred.

For example, the monoclonal antibody can be used in the diagnosticmethods and systems disclosed herein where formation of a C-terminalhGPIIb fragment-containing immunoreaction product is desired.

VI. Hybridomas

Hybridomas of the present invention are those characterized as havingthe capacity to produce an anti-GPIIb C-terminal fragment monoclonalantibody as described before.

Representative preferred hybridomas are prepared as described in Example3. Particularly preferred is the hybridoma culture designated PMI-1.

Hybridoma culture PMI-1 has been deposited pursuant to Budapest Treatyrequirements with the American Type Culture Collection (ATCC),Rockville, Md., on May 17, 1989, and was assigned accession number HB9476.

Hybridoma HB 9476 was deposited in a depository affording permanence ofthe deposit and ready accessibility thereto by the public upon theissuance of a patent, under conditions which assure that access to thehybridoma will be available during the pending of the patent applicationto those deemed by the Commissioner to be entitled to such access, andthat all restrictions on the availability to the public of the hybridomaas deposited will be irrevocably removed upon the granting of thepatent. The deposited hybridoma will be maintained by the ATCC for theterm of the patent or 30 years from the date of deposit, whichever islonger, and in all events for at least five years after the date of thelast request for access. All maintenance fees have been paid.

Methods for producing hybridomas secreting antibody molecules having adesired immunospecificity, i.e., having the ability to immunoreact witha particular protein, an identifiable epitope on a particular proteinand/or a polypeptide, are well known in the art and are describedfurther herein. Particularly applicable is the hybridoma technologydescribed by Niman et al ., Proc. Natl. Acad. Sci. USA, 80:4949-4953(1983), and by Galfre et al., Meth. Enzymol., 73:3-46 (1981), whichdescriptions are incorporated herein by reference.

Other methods of producing a monoclonal antibody, a hybridoma cell, or ahybridoma cell culture are also well known. See, for example, the methodof isolating monoclonal antibodies from an immunological repertoire asdescribed by Sastry, et al., Proc. Natl. Acad. Sci., 86:5728-5732(1989); and Huse et al., Science, 246:1275-1281 (1989).

Also contemplated by this invention is the hybridoma cell, and culturescontaining a hybridoma cell that produce a monoclonal antibody of thisinvention.

VII. Diagnostic Systems

A diagnostic system of the present invention in kit form includes, in anamount sufficient to perform at least one assay, a compositioncontaining polyclonal or monoclonal antibody of this invention orfragments thereof, as a separately packaged reagent. Instructions foruse of the packaged reagent are also typically included.

"Instructions for use" typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like. Also included, in one formor another, may be charts, graphs and the like that demonstratepredetermined concentration levels correlating specific physiologicalconditions to thrombotic events.

A diagnostic system is contemplated for assaying for the presence, andpreferably amount, of C-terminal hGPIIb fragment in a body fluid sample,such as blood, plasma or urine according to the diagnostic methodsdescribed herein.

Preferably, the antibody is present as a monoclonal antibodycomposition, comprising a monoclonal antibody as described herein.

A diagnostic system of the present invention typically also includes alabel or indicating means capable of signaling the formation of aspecifically bound complex containing an antibody molecule of thepresent invention.

As used herein, the terms "label" and "indicating means" in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. Any label or indicatingmeans can be linked to or incorporated in an antibody molecule that ispart of an antibody or monoclonal antibody composition of the presentinvention, or used separately, and those atoms or molecules can be usedalone or in conjunction with additional reagents. Such labels arethemselves well-known in clinical diagnostic chemistry and constitute apart of this invention only insofar as they are utilized with otherwisenovel methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturing them to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyante (FITC),5-dimethylamine-l-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca"Immunofluorescence Analysis", in Antibody As A Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982) which isincorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such ashorseradish peroxidase (HRO), glucose oxidase, or the like. In suchcases where the principal indicating group is an enzyme such as HRP orglucose oxidase, additional reagents are required to visualize the factthat a receptor-ligand complex (immunoreactant) has formed. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2'-azino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

Radioactive elements are also useful labeling agents and are usedillustratively herein. An exemplary radiolabelling agent is aradioactive element that produces gamma ray emissions. Elements whichthemselves emit gamma rays, such as ¹²⁴ I, ¹²⁵ I, ¹²⁸ I, ¹³² I and ⁵¹ Crrepresent one class of gamma ray emission-producing radioactive elementindicating groups. Particularly preferred is ¹²⁵ I. Another group ofuseful labelling means are those elements such as ¹¹ C, ¹⁸ F, ¹⁵ O and¹³ N which themselves emit positrons. The positrons so emitted producegamma rays upon encounters with electrons present in the animal's body.Also useful is a beta emitter, such as ¹¹¹ indium of ³ H.

The linking of labels, i.e., labeling of, polypeptides and proteins iswell known in the art. For instance, antibody molecules produced by ahybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46(1981). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable. See, forexample, Avrameas, et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23(1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No.4,493,795.

The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A "specific binding agent" is amolecular entity capable of selectively binding a reagent species of thepresent invention but is not itself an antibody molecule of the presentinvention. Exemplary specific binding agents are antibody molecules,complement proteins or fragments thereof, protein A and the like.Preferably, the specific binding agent can bind the antibody molecule ofthis invention when it is present as part of a complex.

In preferred embodiments the specific binding agent is labeled. However,when the diagnostic system includes a specific binding agent that is notlabeled, the agent is typically used as an amplifying means or reagent.In these embodiments, the labeled specific binding agent is capable ofspecifically binding the amplifying means when the amplifying means isbound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an "ELISA"format to detect, for example, the presence or quantity of C-terminalhGPIIb fragment in a body fluid sample such as serum, plasma or urine."ELISA" refers to an enzyme-linked immunosorbent assay that employs anantibody or antigen bound to a solid phase and an enzyme-antigen orenzyme-antibody conjugate to detect and quantify the amount of anantigen present in a sample. A description of the ELISA technique isfound in Chapter 22 of the 4th Edition of Basic and Clinical Immunologyby D. P. Sites et al., published by Lange Medical Publications of LosAltos, Calif. in 1982 and in U.S. Pat. Nos. 3,654,090; 3,850,752; and4,016,043, which are all incorporated herein by reference. An exemplaryELISA protocol is described in Example 4.

Thus, in preferred embodiments, the antibody or antigen reagentcomponent can be affixed (operatively linked) to a solid matrix to forma solid support that is separately packaged in the subject diagnosticsystems. The reagent is typically affixed to the solid matrix byadsorption from an aqueous medium, although other modes of affixation,well known to those skilled in the art, can be used. Exemplaryaffixation methods are described in Example 4.

Particularly preferred are embodiments suitable for competition ELISAassays wherein the antibody is in the liquid phase together with asample containing an unknown amount of C-terminal hGPIIb fragment andthe antigen is in the solid phase in an amount sufficient to competewith liquid phase antigen for immunoreaction with the liquid phaseantibody. In this embodiment, which is described in Example 4, theantigen can be hGPIIb analog, C-terminal hGPIIb fragment, or isolatedGPIIb-IIIa.

Useful solid matrices are well known in the art. Such materials includethe cross-linked dextran available under the trademark SEPHADEX fromPharmacia Fine Chemicals (Piscataway, N.J.); agarose; polystyrene beadsabout 1 micron to about 5 millimeters in diameter available from AbbottLaboratories of North Chicago, Ill.; polyvinyl chloride, polystyrene,cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such assheets, strips or paddles; or tubes, plates or the wells of a microtiterplate such as those made from polystyrene or polyvinylchloride.

Thus, in particularly preferred embodiments, a diagnostic kit furthercontains, in a separate package, an antigen as described above for usein a the competitive ELISA assay in the form of a solid phase antigen asdefined above.

The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microtiter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

The packages discussed herein in relation to diagnostic systems arethose customarily utilized in diagnostic systems. Such packages includeglass and plastic (e.g., polyethylene, polypropylene and polycarbonate)bottles, vials, plastic and plastic-foil laminated envelopes and thelike.

VIII. Assay Methods The present invention contemplates any diagnosticmethod that results in detecting C-terminal hGPIIb fragment in a bodyfluid sample using an hGPIIb analog, a substantially isolated C-terminalhGPIIb fragment or an antibody of this invention as a reagent to form animmunoreaction product whose amount relates, either directly orindirectly, to the presence, and preferably amount, of C-terminal hGPIIbfragment in the sample.

Those skilled in the art will understand that there are numerous wellknown clinical diagnostic chemistry procedures in which animmunochemical reagent of this invention can be used to form animmunoreaction product whose amount relates to the amount of C-terminalhGPIIb fragment present in a body sample. Thus, while exemplary assaymethods are described herein, the invention is not so limited.

Various heterogeneous and homogeneous protocols, either competitive ornoncompetitive, can be employed in performing an assay method of thisinvention.

Generally, to detect the presence of a C-terminal hGPIIb fragment in apatient, an aliquot (i.e., a predetermined amount) of a body fluidsample, such as urine or a vascular fluid, namely blood, plasma or serumfrom the patient is contacted by admixture (admixed), with an antibodycomposition of the present invention to form an immunoreactionadmixture. The admixture is then maintained under biological assayconditions for a period of time sufficient for the C-terminal hGPIIbfragment present in the sample to immunoreact with (immunologicallybind) a portion of the antibody combining sites present in the antibodycomposition to form a C-terminal hGPIIb fragment-antibody moleculeimmunoreaction product (immunocomplex). The complex can then be detectedas described herein. The presence of the complex is indicative ofC-terminal hGPIIb fragment in the sample.

Maintenance time periods sufficient for immunoreaction are well knownand are typically from about 10 minutes to about 16-20 hours at atemperature of about 4° C. to about 45° C., with the time andtemperature typically being inversely related. For example, longermaintenance times are utilized at lower temperatures, such as 16 hoursat 4° C., and shorter times for higher temperatures, such as 1 hour atroom temperature.

Biological assay conditions are those that maintain the biologicalactivity of the immunochemical reagents of this invention and theC-terminal hGPIIb fragment sought to be assayed such that the reagentsretain their ability to form an immunoreaction product. Those conditionsinclude a temperature range of about 4° C. to about 45° C., a pH valueof about 5 to about 9 and an ionic strength varying from that ofdistilled water to that of about one molar sodium chloride. Methods foroptimizing such maintenance time periods and biological assay conditionsare well known in the art.

Preferred maintenance times and assay conditions sufficient forimmunoreaction to occur are described in Example 4.

According to the discoveries described herein, thrombosis is accompaniedby the cleavage of the carboxy terminus of hGPIIb by indigenousproteases, to form soluble C-terminal hGPIIb fragment in body fluidsamples. Thus, in one embodiment, the detection of C-terminal hGPIIbfragment in a body sample is utilized as a means to monitor the presenceof an in vivo thrombic event in a patient (thrombosis) using an antibodymolecule specific for the C-terminal hGPIIb fragment as disclosedherein.

In preferred embodiments, immunoassay of C-terminal hGPIIb fragment isconducted using a cell free body fluid sample. By cell free is meantthat the sample does not contain detectable amounts of cells, tissue orother macroscopic biological materials normally present in a body fluidsuch as blood. A body fluid sample typically contains cells as a normalcomponent, or as a contaminant, and can be rendered cell-free by avariety of biochemical procedures including centrifugation, filtrationand chromatography, so long as the retained fluid sample containssubstantially all of the soluble protein initially present in the sampleprior to removal of the cells. Particularly preferred are centrifugationsteps such as described in Example 4.

Thus a body fluid sample, such as blood, known to contain both plateletshaving GPIIb-IIIa thereon and soluble C-terminal hGPIIb fragment, ispreferably treated, prior to admixing, to a manipulation that results informing a cell-free body fluid sample, ie, separating the cells presentin the sample away from the body fluid sample to form a cell-free bodyfluid sample. An aliquot of the cell-free body fluid sample is thenadmixed with an antibody of the invention to form an immunoreactionadmixture.

Alternatively, the separation of C-terminal hGPIIb fragment from cellshaving GPIIb, such as platelets, may be accomplished at other stages ofthe assay methods disclosed herein. For example, the immunoreactionproduct containing C-terminal hGPIIb fragment can be separated fromplatelets present in the body fluid sample by centrifugation after themaintenance step under conditions that separate cells, i.e., platelets,from protein complexes such as an immunocomplex between C-terminalhGPIIb fragment and an antibody of this invention.

Determining the presence or amount of C-terminal hGPIIbfragment-containing immunoreaction product formed by the abovemaintenance step, either directly or indirectly, can be accomplished byassay techniques well known in the art, and typically depend on the typeof indicating means used.

In a preferred competition assay method, the immunoreaction admixturedescribed above further contains a solid phase having affixed thereto asolid phase antigen comprising a hGPIIb analog or a polypeptide havingan amino acid residue sequence that includes the sequence of aC-terminal hGPIIb fragment of this invention. Thus, in this embodiment,the assay comprises the steps of:

(a) admixing a body fluid sample with 1) an antibody composition of thisinvention and 2) a solid support having affixed thereto (operativelylinked) a hGPIIb analog or a polypeptide having an amino acid residuesequence that includes the sequence of a C-terminal hGPIIb fragment ofthis invention, or both, to form an immunoreaction admixture having botha liquid phase and a solid phase;

(b) maintaining said immunoreaction admixture under biological assayconditions for a time period sufficient to form an immunoreactionproduct in the solid phase; and

(c) detecting the presence, and preferably amount, of the immunoreactionproduct formed in the solid phase in step (b), and thereby the amount ofpresence/amount of C-terminal hGPIIb fragment in the body fluid sample.

Where an hGPIIb analog is used in the solid phase, the antibodycomposition contains antibody molecules that immunoreact with the hGPIIbanalog. Preferably, the solid phase antigen is isolated GPIIb-IIIa,prepared as described in Example 1b, or substantially isolatedC-terminal hGPIIb fragment, prepared as described in Example 1c.

Preferably, the body fluid sample is a cell free body fluid sample suchas urine or platelet poor plasma.

More preferably, detecting in step (c) is performed by the steps of:

(i) admixing the immunoreaction product formed in step (b) with anindicating means to form a second reaction admixture;

(ii) maintaining the second reaction admixture for a time periodsufficient for said indication means to bind the immunoreaction productformed in step (b) and form a second reaction product; and, (iii)determining the presence and/or amount of indicating means in the secondreaction product, and thereby the presence of the immunoreaction productformed in step (b). Particularly preferred is the use of a labeledsecond antibody, immunospecific for the first antibody, as theindicating means, and preferably the label is horseradish peroxidase. Inone embodiment, it is particularly preferred to use (1) isolatedGPIIb-IIIa as the solid phase antigen, (2) PMI-1 monoclonal antibody inthe antibody composition, and (3) goat anti-mouse IgG antibodies labeledwith horseradish peroxidase as the indicating means. Exemplary is thecompetition ELISA format described in Example 4.

In another competition assay format the immunoreaction admixturecontains (1) a body fluid sample, preferably cell free, (2) an antibodyof this invention and (3) a labeled hGPIIb analog or labeled C-terminalhGPIIb fragment, wherein the antibody is present in the solid phase,being affixed to a solid support, to form a liquid and a solid phase. Inthis embodiment, the admixed body fluid sample competes with the labeledreagent for immunoreaction with the solid phase antibody to form a solidphase immunoreaction product. Thereafter, the detection of label in thesolid phase correlates with the amount of C-terminal hGPIIb fragment inthe admixed fluid sample.

The following examples are intended to illustrate, but not limit, thepresent invention.

Examples 1. Preparation of Substantially Isolated C-Terminal hGPIIbFragment a. Platelet Isolation

One unit of whole human blood was collected in ACD (0.065M citric acid,0.085M sodium citrate, 2% dextrose) containing hirudin (Sigma ChemicalCo., St. Louis, Mo.) at a final concentration of 0.06 units permilliliter (U/ml) and centrifuged for 15 minutes (min) at 120×g. Theresulting supernatant, designated platelet-rich plasma (PRP), wasrecovered, isolated and further centrifuged for 15 min at 1200×g to forma pellet of isolated platelets. The supernatant formed was collected anddesignated platelet-poor plasma (PPP).

b. GPIIb-IIIa Isolation from Platelets

A platelet pellet prepared as in Example la, was resuspended in 5 ml TBS(0.15M NaCl, 0.2M Tris, pH 7.4, 5×10⁻⁴ M CaCl₂, 10⁻⁵ M leupeptin) andsonicated on ice for 10 min at a maximum setting using a Model W-375sonicator (Heat Systems Ultrasonics, Plainview, NY). The sonicatedsuspension was twice frozen and thawed using a dry ice-methanol ice bathand stored at minus 20 degrees C (°C.). The frozen-thawed plateletsonicate was layered on top of 5 ml of a sucrose solution (40% v/v inTBS), and centrifuged at 4 degrees C for one hour at 38,000 rotationsper minute (RPM) in a SW41 centrifuge rotor (Beckman Instruments,Fullerton, Calif.) to form a milky colored infranatant. Themilky-infranatant was then recovered and centrifuged at 43,000 RPM in aSW50.1 centrifuge rotor (Beckman) at 4° C. for one hour. The resultingpellet was resuspended in typically 1-2 ml TBS to form a plateletmembrane solution, the protein concentration of which was determined tobe in the range of 10-25 mg/ml, typically using the Bio-Rad ProteinAssay Kit (Bio-Rad, Richmond, Calif.) according to the manufacturer'sinstructions.

The platelet membrane solution was again centrifuged in a SW50.1centrifuge rotor as above and the resulting pellet was resuspended in 2ml of extraction buffer (0.03 M Tris, pH 7.4, 1×10⁻⁵ M leupeptin, 200 mMn-octyl-beta-D-glucopyranoside; Calbiochem-Behring, La Jolla, Calif.).The platelet membrane extract thus formed was admixed thoroughly byvortexing and then maintained at room temperature for 30 min. Theextract was thereafter centrifuged at 45,000 rpm in a SW50.1 centrifugerotor for 1 hour at 4° C. and the platelet membrane extract supernatantthus formed was recovered.

The recovered supernatant was applied to a LKB Ultrogel Aca 34 gelfiltration column (3×97 cm, LKB Instruments, Gaithersburg, Md.) that hadbeen equilibrated with 1 liter of column buffer (0.03 M Tris, pH 7.4,0.1 mM CaCl₂, 0.1% n-octyl-beta-D-glucopyranoside) and 5 ml fractionswere collected from the resulting column effluent. The optical densityat 280 nanometers of each fraction was determined and fractions aroundthe several peaks were combined to form a pool for each peak. Samplesfrom each pool were analyzed by electrophoresis in 6% polyacrylamideslab gels using the reducing buffers and procedures described byLaemmli, Nature (London), 227:680-685 (1970), and low molecular weightprotein standards ranging in size from 14.4 kilodaltons (kDa) to 92.5kDa (Bio-Rad, Richmond, Calif.). The pool containing predominantly twoprotein species having molecular weights corresponding to GPIIb andGPIIIa, i.e., 120 kDa and 100 kDa, respectively was recovered and isdesignated as a composition containing isolated GPIIb-IIIa. The proteinconcentration of the isolated GPIIb-IIIa containing-composition thusprepared was typically determined using the Bio-Rad Protein Assay Kitsto be in the range of 0.3 to 0.8 mg/ml.

c. Isolation of Carboxy-Terminal Fragment of hGPIIb from Platelets (1)Digestion with Proteases

A platelet pellet prepared as in Example la was resuspended in Tyrode'ssolution (Sigma; 137 mM NaCl, 2 mM KCl, 12 mM NaHCO₃, 0.3 mM NaH₂ PO₄, 2mM CaCl₂, 1 mM MgCl₂, 5.5 mM glucose, 5 mM HEPES, pH 7.4) supplementedwith 100 nM PGE1 (Sigma; prostaglandin inhibitor of activation), apyrase(Sigma)(primary suspension contained 7.5 ug/ml of PRP; washes contained25 ug/ml), and BSA, 3.5 mg/ml. After two centrifugations in supplementedTyrode's, the platelet concentration was adjusted to 1×10⁹ per ml insupplemented Tyrode's. Alpha-chymotrypsin (Sigma) was added to produce afinal concentration of 0.75 mg/ml and the solution was incubated for 60min at 37 degrees C. The reaction was then stopped by adding a 200 mMstock solution of phenylmethylsulfonyl fluoride (PMSF) to form a finalconcentration of 1.0 mM PMSF in the platelet suspension.

During the time course of the 1 hour chymotrypsin incubation of theplatelet suspension, aliquots of 0.2 ml containing 2×10⁸ platelets weretaken and processed further. A first aliquot (0.2 ml) of plateletsuspension was centrifuged at 15,000×g for 5 min at room temperature toform a supernatant and a pellet. The supernatant was collected to form asupernatant sample. The pellet was resuspended in 0.2 ml of supplementedTyrode's containing 1 mM PMSF to form a resuspended pellet sample foruse in the ELISA assay described in Example 4b. A second aliquot (0.2ml) of platelet suspension was used directly as a total sample in theELISA assay of Example 4b. The presence, if any, of C-terminal hGPIIbfragment was then measured in the three samples (supernatant sample,resuspended pellet sample, and total sample extract) using thecompetition ELISA assay described in Example 4b.

The results are shown in FIG. 1, and indicate the levels of the PMI-1epitope (FIG. 1A) and the levels of the TAB epitope (FIG. 1/B) in thethree fractions. Monoclonal antibody PMI-1 immunoreacts with the PMI-1epitope on hGPIIb defined by the polypeptide IHPAHHK (SEQ ID NO 3 fromresidue 194 to residue 200). Monoclonal antibody TAB immunoreacts withthe TAB epitope located on a portion of hGPIIb other than the C-terminalhGPIIb fragment, because the material immunoreactive with TAB does notbecome released into the supernatant after chymotrypsin digestion.

(2) Isolation of C-Terminal hGPIIb Fragment

Isolated PMI-1 antibody molecules prepared as described in Example 3c,are covalently linked to Sepharose beads using cyanogenbromide-activated Sepharose (Pharmacia) according to the manufacturer'sinstructions to form PMI-1 Sepharose having about 20 mg of antibodyprotein per ml of packed beads.

Five ml of chymotrypsin digested platelet suspension, prepared as inExample 1c(1), is admixed with 5 ml of packed PMI-1 Sepharose beads andthe resulting admixture is maintained at 4° C. for 12-18 hours to allowany C-terminal hGPIIb fragment in the suspension to bind to the PMI-1Sepharose. The PMI-1 Sepharose is then transferred to a columncontaining a scintered glass support, and washed with 25 ml of washbuffer (described in Example 4), also containing 50 mM octylglucoside.Thereafter wash buffer containing 50 mM octylglucoside is added to thecolumn and 1.25 ml fractions are collected as the added buffer comes off(elutes from) the column to form eluted fractions.

Fifty microliters (ul) of each eluted fraction is then analyzed bysodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis(SDS-PAGE) using a 10-20% acrylamide gel gradient containing 3% SDS and5% 2-mercaptoethanol according to the methods of Laemmli, Nature,227:680-685 (1970). Proteins contained in the eluted fractions soanalyzed are transferred from the SDS-PAGE gels to nitrocellulose andvisualized by autoradiography according to the method of Johnson et al,Gen. Anal. Techn., 1:3-8 (1984) using ¹²⁵ I-labeled PMI-1. The apparentmolecular weight of the visualized proteins is determined by comparisonto protein molecular weight markers also analyzed on the gel. Allmolecular weights are in units of one thousand daltons.

Fractions containing eluted proteins that reacted with PMI-1 antibodyare collected and pooled to form an isolated C-terminal hGPIIbfragment-containing solution, i.e., substantially isolated hGPIIbfragment. The isolated protein exhibited an apparent weight of about3,900 daltons when analyzed by SDS-PAGE, and constituted the majorprotein component in the isolated fragment-containing solution.

The amino acid residue sequence of the C-terminal hGPIIb fragmentidentified herein after in vitro proteolysis of platelets is shown inSEQ ID NO 3 from residue 173 to residue 200.

2. Preparation of Polypeptide Analogs

Based on the amino acid residue sequence deduced from the nucleotidesequence of hGPIIb, polypeptides corresponding to the various hGPIIbC-terminal regions utilized herein are chemically synthesized on anApplied Biosystems Model 430A Peptide Synthesizer using the symmetricalanhydride method of Hagenmaier, et al., Hoppe-Sevler's Z. Phisiol.Chem., 353:1973 (1982).

The amino acid residue sequences of the various polypeptide analogssynthesized are shown in Table 1.

3. Antibody Production a. Preparation of Immunogen

The term "immunogen", as used herein, describes an entity that inducesantibody production in the host animal. In some instances, the antigenand immunogen are the same entity, while in other instances, the twoentities are different. Immunogens used to elicit antibodies of thisinvention are whole platelets, platelet membranes, isolated GPIIb-IIIa,isolated C-terminal hGPIIb fragment or hGPIIb polypeptide analogs asdescribed above.

Polyclonal anti-peptide antibodies are produced by immunizing rabbitswith a peptide prepared in Example 2 above and coupled to carrierprotein. Coupling of the peptide with glutaraldehyde to thyroglobulin asantigenic carrier (Bovine Type I, Sigma Chemical Co., St. Louis, Mo.) isconducted as described by J. G. Dockray, Regulatory Peptides, 1:169-186(1980), which is hereby incorporated by reference to form thyroglobulincoupled peptide.

b. Preparation of Polyclonal Antibody Compositions

Immunizations to produce a polyclonal antibody composition are doneaccording to standard protocols. Briefly, a rabbit is immunizedintradermally at one week intervals with 400 micrograms (ug) ofimmunogen consisting of thyroglobulin coupled peptide at 3 mg/ml. Theimmunogen is diluted 1:1 in Complete Freund's Adjuvant for the firstimmunization, in Incomplete Freund's Adjuvant for the second and thirdimmunizations, and in normal saline for the fourth and fifth.

Injections are administered with 100 ug distributed over 4 sites.Thereafter, the antibody is harvested and tested according to the assayof Example 4a. In that assay, polyclonal antibody produced using apeptide of Table 1 immunoreacts with the carboxy terminal hGPIIbfragment and with the immunizing peptide, but does not immunoreact withthe polypeptide REQNSLDSWGPK (SEQ ID NO 3 from residue 113 to residue124).

C. Preparation of Monoclonal Antibody Compositions

Monoclonal antibodies that immunoreact with C-terminal hGPIIb fragmentwere produced using standard hybridoma technology with exceptions asnoted. Balb C mice were immunized biweekly with 50 ug of plateletmembranes prepared as described by Barber et al., J. Biol. Chem.,245:6357-65 (1970), incorporated herein by reference, emulsifiedalternately in complete and incomplete Freund's adjuvant. Mice were bledfrom the retro-orbital plexus, and sera were screened for antibodies bysolid-phase radioimmunoassay as described in Shadle et al., J. CellBiol., 99:2056-2060 (1984). Three days after the fifth immunization,about 1×10⁸ lymphocytes were isolated from the spleens of mice, admixedinto a suspension and fused with 5×10⁷ P3X63AG8.053 mouse myeloma cellsusing 50% PEG 4000 as the cell fusion promoter. The resultanttransformed (fused) antibody-producing cells (hybridomas) were initiallytransferred to 96-well microtiter plates at a density of about 1×10⁶cells per well and cultured in selective HAT medium.

Tissue culture supernatant appearing to contain viable HAT resistanthybridoma cells after 8 days of culturing were screened in the ELISAassay described in Example 4a for the presence of antibody moleculesthat immunoreact with GPIIb-IIIa prepared as described in Example 1b.Hybridoma cultures were identified that producedGPIIb-IIIa-immunoreacting antibody molecules. The isolated hybridomaswere then subcloned twice at limiting dilutions to provide about 1 cellper well and 24 of the resultant hybridoma cultures were shown to be ofmonoclonal origin on the basis of three criteria: (1) each supernatantwas from a single cell focus and immunoreacted with GPIIb-IIIa in theELISA screen, (2) each supernatant showed a single homogeneous band whenanalyzed by cellulose acetate gel electrophoresis according to themethod described in Monoclonal Antibodies: Principles and Practice, J.W. Goding, ed., Academic Press, Inc., Orlando, Fla., 1983, and (3) eachsupernatant contained a single isotype of immunoglobulin when analyzedusing the Mouse Ig Screening and Isotyping Kit according to theinstructions provided by the manufacturer, Boehringer-MannheimBiochemicals, Indianapolis, Ind..

One monoclonal antibody producing hybridoma, PMI-1, isolated by theabove procedure using an inoculum containing platelet membranes isavailable from the ATCC and has an accession number HB 9476.

A culture of hybridoma cells capable of secreting PMI-1 monoclonalantibodies were introduced into a mouse and the ascites fluid was thenharvested as is well known. PMI-l monoclonal antibody molecules presentin the harvested ascites were isolated using protein-A Sepharose(Pharmacia, Piscataway, N.J.) according to the manufacturer'sinstructions to form isolated PMI-1 antibody molecules.

The protein concentration of isolated antibody molecules in a liquidcomposition was determined, as needed, using the Bio-Rad Protein AssayKit (Bio-Rad, Richmond, Calif.) according to the manufacturer'sinstructions.

To prepare a monoclonal antibody composition containing ¹²⁵ I-labeledantibody molecules, 350 microliters (ul) of PBS (0.15 M NaCl, 0.01Msodium phosphate, pH 7.09) containing 1 milligram per milliliter (mg/ml)of the above isolated PMI-1 antibody molecules were admixed with 40micrograms (ug) of chloramine-T and 1 millicurie (mCi) of carrier-freeNa¹²⁵ I (Amersham, Arlington Heights, Ill.). The resulting admixture wasmaintained for 5 minutes at about 20° C. and then admixed with 20 ul ofa 2 mg/ml sodium metabisulfite solution (2 mg/ml) and 20 ul of apotassium iodide solution. Thereafter, 800 ul of PBS containing 1% BSAwere admixed followed by further admixture of diisopropylfluorophosphateto a final concentration of 10 mM. The resulting admixture wasmaintained for 60 minutes at 22° C. and then dialyzed against PBS. Thespecific activity of the resulting 125I-labeled antibody molecules wasabout 4.5 microcurie (uCi) per ug.

Compositions containing Fab fragments were prepared by digestingisolated PMI-1 antibody molecules with papain (200:1 weight per weightof Ig to papain) for 6 hours at 37° C. following the methods of Mage etal., Methods in Enzymology, 70:142-150 (1980). Undigested Ig and Fcfragments were removed by chromatography on protein A-Sepharose. Theresulting Fab fragments-containing compositions were then ready for use,or were ¹²⁵ I-labeled, as needed, using the same procedures as describedabove for monoclonal antibody compositions.

4. Diagnostic Assays a. ELISA To Screen Monoclonal Antibodies

Antibody molecules contained in hybridoma culture supernatant areexamined for their ability to immunoreact with hGPIIb polypeptideanalogs. Fifty microliters (ul) of coating solution (0.1M NaHCO₃, pH8.0, 0.1% NaN₃) containing 10 ug/ml of polypeptide analogs prepared inExample 2 are admixed into the wells of flat-bottom 96-well microtiterplates (Immulon 2; Dynatech Laboratories, Chantilly, Va.). The platesare then maintained for 60 minutes at 37° C. to permit the GPIIb-IIIa toadsorb onto the walls of the wells. The coating solution is removed byshaking, the wells are rinsed twice with wash buffer (10 mM Tris at pH7.4, 0.05% (v/v) TWEEN-20, 0.15M NaCl, and 200 mg/ml merthiolate), andthen 200 ul of blocking solution 5% bovine serum albumin (BSA;w/v) incoating solution! are admixed into each well (solid support) to blockexcess protein sites.

The wells are maintained for 60 minutes at about 37° C. and then theblocking solution is removed. About 50 ul of hybridoma culturesupernatant diluted 1:1 in dilution buffer consisting of 0.1% (w/v) BSAin wash buffer is added to each well to form an immunoreactionadmixture. The resulting solid/liquid phase immunoreaction admixturesare maintained at room temperature for 60 minutes to permit formation ofa first solid phase-bound immunoreaction product between the solidphase-bound polypeptide analog and admixed antibodies. The solid andliquid phases are then separated, the wells are rinsed twice with washbuffer, and excess liquid is removed by shaking.

Fifty ul of a solution containing horseradish peroxidase labeled goatanti-mouse IgG (Tago Inc., Burlingame, CA), diluted 1:1000 in dilutionbuffer is admixed into each well to form a second solid/liquid phaseimmunoreaction admixture (labeling immunoreaction admixture). The wellsare maintained for 60 minutes at room temperature to permit formation ofa second immunoreaction product between the labeled antibody and anysolid phase-bound antibody of the first immunoreaction product and thenrinsed twice with wash buffer to isolate the solid phase-boundlabel-containing immunoreaction products. Excess liquid is then removedfrom the wells.

Fifty ul of freshly prepared chromogenic substrate solution containing4.0 mg/ml O-phenylenediamine and 0.012% (v/v) hydrogen peroxide in CPbuffer (243 ml of 0.1M citric acid and 250 ml of 0.2M dibasic sodiumphosphate per liter H₂ O, pH 5.0) are then admixed into each well toform a color developing-reaction admixture. After maintaining the colordeveloping-reaction admixture for 10 minutes at about 20° C., 50 ul of2N H₂ SO₄ are admixed into each well to stop the developing-reaction,and the resulting solutions are assayed for absorbance at 490 nanometers(nm) light wavelength using a Model 310 ELISA plate reader (Bio-TekInstruments, Winooski, Vt.).

Antibody molecule compositions are considered to contain antibodymolecules immunoreactive with C-terminal hGPIIb fragment if the measuredabsorbance at 490 nm (A490) is at least 6 times above background i.e.,above about 0.3 optical density units when measured at A490.

b. Competition ELISA Assays to Detect C-Terminal GPIIb Heavy ChainFragment in Body Fluids

Assays to detect C-terminal hGPIIb fragment can be performed on"platelet-poor" plasma, serum or urine samples without concentration.Urine (serum and plasma are used in similar fashion) samples werecentrifuged at 15,000×g for 5 min at room temp, and the resultingsupernatants were collected to form cell free urine samples. Theresulting cell-free urine samples are substantially free of plateletsand therefore any hGPIIb fragment present in the sample is in the formof soluble, cell-free, hGPIIb fragment. Fifty microliters (ul) ofcoating solution (0.1M NaHCO₃, pH 8.0, 0.1% NaN₃) containing 10 ug/ml ofisolated GPIIb-IIIa prepared as described in Example lb were admixedinto the wells of flat-bottom 96-well microtiter plates (Immulon 2;Dynatech Laboratories, Chantilly, Va.). The plates were then maintainedfor 60 minutes at 37° C. to permit the GPIIb-IIIa to adsorb onto thewalls of the wells. The coating solution was removed by shaking and 200ul of blocking solution 5% bovine serum albumin (BSA;w/v) in coatingsolution! were admixed into each well (solid support) to block excessprotein sites.

The wells were maintained for 60 minutes at about 37° C. and then theblocking solution was removed by rinsing the wells thrice with washbuffer (10 mM Tris at pH 7.4, 0.05% (v/v) TWEEN-20, 0.15M NaCl, and 200mg/ml merthiolate). Isolated monoclonal antibody PMI-1 prepared inExample 2c or a control monoclonal antibody (TAB), that immunoreactswith hGPIIb but does not immunoreact with C-terminal hGPIIb fragment,were used in the competition ELISA. TAB was obtained from Dr. R. McEver,University of Texas (San Antonio, Tex.). About 50 ul of monoclonalantibody, diluted 1:1 in incubation buffer consisting of 0.1% (w/v) BSAin wash buffer, was admixed, along with the sample to be tested, to eachwell to form an immunoreaction admixture. The proportions were asfollows:

(a) 20 ul incubation buffer+10 ul of 20 mM EDTA in incubation buffer+20ul of sample prepared in Example 1c(i); and

(b) 50 ul PMI-1 or TAB monoclonal antibody 1:1000 of 4.5 mg/ml IgG(purified on Staph. A columns)!.

One set of immunoreaction admixtures was prepared using a test sample,and a second set of immunoreaction admixtures was prepared in which thesample is replaced with a reference standard comprised of apredetermined amount of either isolated GPIIb-IIIa, if the antibody wasTAB, or polypeptide V43 (PSPSPIHPAHHKRDRRQ), (SEQ ID NO 2) prepared asin Example 2, if the antibody was PMI-1.

Each immunoreaction admixture was incubated overnight at roomtemperature to permit formation of a first solid phase-boundimmunoreaction product containing the solid phase-bound GPIIb-IIIa andthe admixed antibodies.

The solid and liquid phases were then separated, the wells were rinsedthrice with washing buffer, and excess liquid was removed by shaking.Fifty ul of a solution containing horseradish peroxidase labeled goatanti-mouse IgG (Tago Inc., Burlingame, Calif.), diluted 1:1000 inincubation buffer was admixed into each well to form a secondsolid/liquid phase immunoreaction admixture (labeling immunoreactionadmixture). The wells were maintained for 60 minutes at room temperatureto permit formation of a second immunoreaction product between thelabeled antibody and any solid phase-bound antibody of the firstimmunoreaction product and then rinsed thrice with wash buffer toisolate the solid phase-bound label-containing immunoreaction products.Excess liquid was then removed from the wells.

Fifty ul of freshly prepared chromogenic substrate solution containing4.0 mg/ml O-phenylenediamine and 0.012% (v/v) hydrogen peroxide in CPbuffer (243 ml of 0.1M citric acid and 250 ml of 0.2 M dibasic sodiumphosphate per liter H₂ O, pH 5.0) were then admixed into each well toform a color developing-reaction admixture. After maintaining the colordeveloping-reaction admixture for 10 minutes at about 20° C., 50 ul of2N H₂ SO₄ were admixed into each well to stop the developing-reaction,and the resulting solutions were assayed for absorbance at 490nanometers (nm) light wavelength using a Model 310 ELISA plate reader(Bio-Tek Instruments, Winooski, Vt.).

Absorbance measurements obtained using standard were plotted againstmicromolar (uM) concentration of standard in the immunoreactionadmixture to produce a standard curve. Absorbance measurements obtainedusing samples were compared to the standard curve to produce a valueexpressed as equivalents of standard.

FIG. 1 shows results of the above competition ELISA using samplescontaining C-terminal hGPIIb fragment that were formed as described inExample lc(l). The data is expressed as equivalents of standard (inmicromoles) detectable in the sample during the time course of thechymotrypsin digestion. Using polypeptide V43 as a standard incombination with antibody PMI-1, antigenic material that corresponds toC-terminal hGPIIb fragment was released into the supernatant during thecourse of the digest (FIG. 1A). In contrast, no detectable antigens werereleased in the supernatant that immunoreact with the TAB antibody (FIG.1B).

Urine samples from a normal donor in the form of cell free urine samplesas described above were tested in the above competition ELISA usingvarious dilutions of urine in incubation buffer from undiluted to 1 parturine to 7 parts buffer (1:8). Equivalents of peptide V43 detectable inthe urine using PMI-1 were 1.5 uM at 1:8, 3.8 uM at 1:4 and 1.5 uM at1:2. From the data, a normalized value of C-terminal hGPIIb fragment inundiluted urine was 14.4 uM for the normal donor's urine. Thus levels ofC-terminal hGPIIb fragment in urine that are significantly greater thanabout 15 uM, such as a 10% increase over normal values, is indicative ofthromotic activity above normal levels.

Anti-peptide antibodies prepared as described in Example 3c using thepolypeptides shown in Table 1 are used in the competition ELISA in placeof monoclonal antibody PMI-1, and using the antibody's correspondingpolypeptide rather than isolated GPIIb-IIIa as the solid-phrase antigen.In this assay, samples containing competing antigen, i.e., C-terminalhGPIIb fragment, or containing standard comprised of the samecorresponding peptide, are measured as above for the presence and amountof C-terminal hGPIIb fragment. By this competition ELISA assay,anti-peptide antibodies that immunoreact with a polypeptide shown inTable 2 are shown to also immunoreact with isolated GPIIb/IIIa and withC-terminal hGPIIb fragment prepared in Example 1c(2) Although thepresent invention has now been described in terms of certain preferredembodiments, and exemplified with respect thereto, one skilled in theart will readily appreciate that various modifications, changes,omissions and substitutions may be made without departing from thespirit thereof. It is intended, therefore, that the present invention belimited solely by the scope of the following claims.

Sequence Listing

SEQ ID NO 1 illustrates the nucleotide and corresponding amino acidresidue sequence of a cDNA that codes for a portion of GPIIb heavy chain(hGPIIb) wherein the nucleotide sequence is shown from left to right andin the direction of 5' terminus to 3' terminus using the single letternucleotide base code represented as an uninterrupted linear series ofbases from base 1 to base 600. The amino acid residue sequence is shownfrom left to right and in the direction from amino-terminus tocarboxy-terminus using the single letter amino acid residue coderepresented as an uninterrupted linear series of residues from residue 1(G) at the amino-terminus to residue 200 (K) at the carboxy-terminus.

The reading frame is indicated by the placement of the deduced aminoacid residue sequence below the nucleotide sequence such that the singleletter that represents each amino acid residue is located below thefirst base in the corresponding codon.

The nucleotide base sequence shown from base 1 through base 600 in SEQID NO 1 corresponds to the nucleotide sequence shown in FIG. 2 of Ponczet al., J. Biol. Chem., 262:8476-8482(1987), from base 2060 to base2659. The amino acid residue sequence from residue 1 to residue 200shown in SEQ ID NO 3 corresponds to the sequence shown in FIG. 2 ofPoncz et al., supra, from residue 656 to residue 855. ##STR1##

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 600 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..600                                                          (D) OTHER INFORMATION: /standard_name= "Portion of GPIIb                      heavy chain"                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGCGCCCACTACATGCGGGCCCTAAGCAATGTCGAGGGCTTTGAGAGA48                            GlyAlaHisTyrMetArgAlaLeuSerAsnValGluGlyPheGluArg                              151015                                                                        CTCATCTGTAATCAGAAGAAGGAGAATGAGACCAGGGTGGTGCTGTGT96                            LeuIleCysAsnGlnLysLysGluAsnGluThrArgValValLeuCys                              202530                                                                        GAGCTGGGCAACCCCATGAAGAAGAACGCCCAGATAGGAATCGCGATG144                           GluLeuGlyAsnProMetLysLysAsnAlaGlnIleGlyIleAlaMet                              354045                                                                        TTGGTGAGCGTGGGGAATCTGGAAGAGGCTGGGGAGTCTGTGTCCTTC192                           LeuValSerValGlyAsnLeuGluGluAlaGlyGluSerValSerPhe                              505560                                                                        CAGCTGCAGATACGGAGCAAGAACAGCCAGAATCCAAACAGCAAGATT240                           GlnLeuGlnIleArgSerLysAsnSerGlnAsnProAsnSerLysIle                              65707580                                                                      GTGCTGCTGGACGTGCCGGTCCGGGCAGAGGCCCAAGTGGAGCTGCGA288                           ValLeuLeuAspValProValArgAlaGluAlaGlnValGluLeuArg                              859095                                                                        GGGAACTCCTTTCCAGCCTCCCTGGTGGTGGCAGCAGAAGAAGGTGAG336                           GlyAsnSerPheProAlaSerLeuValValAlaAlaGluGluGlyGlu                              100105110                                                                     AGGGAGCAGAACAGCTTGGACAGCTGGGGACCCAAAGTGGAGCACACC384                           ArgGluGlnAsnSerLeuAspSerTrpGlyProLysValGluHisThr                              115120125                                                                     TATGAGCTCCACAACACTGGCCCTGGGACTGTGAATGGTCTTCACCTC432                           TyrGluLeuHisAsnThrGlyProGlyThrValAsnGlyLeuHisLeu                              130135140                                                                     AGCATCCACCTTCCGGGACAGTCCCAGCCCTCCGACCTGCTCTACATC480                           SerIleHisLeuProGlyGlnSerGlnProSerAspLeuLeuTyrIle                              145150155160                                                                  CTGGATATACAGCCCCAGGGGGCGCTTCAGTGCTTCCCACAGCCTCCT528                           LeuAspIleGlnProGlnGlyAlaLeuGlnCysPheProGlnProPro                              165170175                                                                     GTCAATCCTCTCAAGGTGGACTGGGGGCTGCCCATCCCCAGCCCCTCC576                           ValAsnProLeuLysValAspTrpGlyLeuProIleProSerProSer                              180185190                                                                     CCCATTCACCCGGCCCATCACAAG600                                                   ProIleHisProAlaHisHisLys                                                      195200                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ProSerProSerProIleHisProAlaHisHisLysArgAspArgArg                              151015                                                                        Gln                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 200 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GlyAlaHisTyrMetArgAlaLeuSerAsnValGluGlyPheGluArg                              151015                                                                        LeuIleCysAsnGlnLysLysGluAsnGluThrArgValValLeuCys                              202530                                                                        GluLeuGlyAsnProMetLysLysAsnAlaGlnIleGlyIleAlaMet                              354045                                                                        LeuValSerValGlyAsnLeuGluGluAlaGlyGluSerValSerPhe                              505560                                                                        GlnLeuGlnIleArgSerLysAsnSerGlnAsnProAsnSerLysIle                              65707580                                                                      ValLeuLeuAspValProValArgAlaGluAlaGlnValGluLeuArg                              859095                                                                        GlyAsnSerPheProAlaSerLeuValValAlaAlaGluGluGlyGlu                              100105110                                                                     ArgGluGlnAsnSerLeuAspSerTrpGlyProLysValGluHisThr                              115120125                                                                     TyrGluLeuHisAsnThrGlyProGlyThrValAsnGlyLeuHisLeu                              130135140                                                                     SerIleHisLeuProGlyGlnSerGlnProSerAspLeuLeuTyrIle                              145150155160                                                                  LeuAspIleGlnProGlnGlyAlaLeuGlnCysPheProGlnProPro                              165170175                                                                     ValAsnProLeuLysValAspTrpGlyLeuProIleProSerProSer                              180185190                                                                     ProIleHisProAlaHisHisLys                                                      195200                                                                        __________________________________________________________________________

What is claimed is:
 1. A hGPIIb analog consisting of a polypeptidehaving an amino acid residue sequence that corresponds to the sequenceof hGPIIb shown in SEQ ID NO 3 from residue 1 to residue
 200. 2. Asubstantially isolated C-terminal hGPIIb polypeptide that has amolecular weight of about 3,900 daltons and comprises an amino acidresidue sequence shown in SEQ ID NO 3 from residue 173 to
 200. 3. AhGPIIb analog consisting of a polypeptide having an amino acid residuesequence that corresponds to the sequence of hGPIIb selected from thegroup consisting of the sequence shown in SEQ ID NO 3 from residues 173to 181, from residues 179 to 189, from residues 187 to 200, and fromresidues 194 to 200.